INFECTION AND IMMUNITY, Aug. 1983, p. 657-6650019-9567/83/080657-09$02.00/0Copyright C 1983, American Society for Microbiology

Vol. 41, No. 2

Preferential Immune Response to Virion SurfaceGlycoproteins by Caprine Arthritis-Encephalitis Virus-Infected

GoatstGAYLE C. JOHNSON,f* ANTHONY F. BARBET, PAULA KLEVJER-ANDERSON, AND TRAVIS C.

McGUIREDepartment of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington

99164

Received 22 November 1982/Accepted 11 May 1983

Six months after inoculation with caprine arthritis-encephalitis virus, the serumand synovial fluid of virus-infected goats had antibodies to [35S]methionine-labeled viral proteins with apparent molecular weights of 125,000, 90,000, 28,000,and 15,000. The 125,000-, 90,000-, and 15,000-molecular-weight methionine-labeled proteins were identified as virion surface glycoproteins by lactoperoxidaseiodination and galactose oxidase-boro[3H]hydride reduction labeling techniques.Radioimmunoassay antibody titers to purified p28, the most abundant viralstructural protein, averaged 1:182 in synovial fluid and 1:67 in serum 6 monthsafter inoculation. High dilutions of serum and synovial fluid reacted with gp9O andgp125 electroblotted onto nitrocellulose paper from polyacrylamide gels. Anti-gp9O activity was detected at dilutions with an immunoglobulin G content of 0.02to 11 ,ug, whereas antibody to p28, when detectable on Western blots, was presentin samples with an immunoglobulin G content of 0.1 to 2 mg, representing 100- to1,000-fold-greater titers of antibody to the surface glycoprotein. Synovial fluidsoften contained more anti-gp90 antibody than did sera. Immunoprecipitation oflactoperoxidase-iodinated virus confirmed the presence of high antibody titers tothe two virion surface glycoproteins. Because antiviral gp90 and gp125 antibody isabundant in the synovial fluid of infected goats, it probably contributes to the highimmunoglobulin Gl concentrations seen at this site 6 months after caprinearthritis-encephalitis virus infection.

Caprine arthritis-encephalitis virus (CAEV), aretrovirus, induces chronic, progressive jointdisease in experimentally infected goats (2, 12,13) and may be isolated from synoviocyte ex-plants of goats with a similar spontaneous dis-ease (13, 14). The presence of follicle-like lym-phoid nodules and focally numerous plasmacells in CAEV-infected synovial tissues (2, 12,14) is correlated immunologically with a peaktwo- to fivefold increase in synovial fluidimmunoglobulin Gl (IgGl) over serum concen-trations, representing 15 to 25 times the concen-tration of IgGl found in synovial fluid of controlgoats (25, 35). After a period of irregular fluctua-tions, synovial fluid IgG declines to a level 40 to70% of that in serum, similar to the relativeconcentrations found in rheumatoid arthritis(47). The nonsuppurative synovitis of CAEVinfection fulfills many of the diagnostic criteria

t Paper no. 6427 from the Agricultural Research Center ofWashington State University.

t Present address: Department of Veterinary Pathobiology,Ohio State University, Columbus, OH 43210.

for rheumatoid arthritis (42), a disease of specu-lated viral etiology (3, 18, 39).Other retroviral infections are commonly as-

sociated with the development of immune-sys-tem-mediated diseases involving virus-antibodycomplexes (38), implying a substantial commit-ment of the host's immune system to the produc-tion of antiviral antibody. In murine leukemiavirus infection, this response is directed in greatpart toward viral glycoproteins expressed on thevirion and infected cell surface (15, 26), and it isnotable that elevated anti-gp7O activity has beenfound in the serum of spontaneously arthriticMRL/1 mice (23). Even in diseases where therelative degree of antibody production to vari-ous viral antigens has not been identified, viralsurface glycoproteins (or antigens responsiblefor virus neutralization) appear important in theantiviral immune response, with or without pro-tection from the disease (4, 29, 36). Becauseviral inoculation initiates CAEV synovitis, andbecause virus remains present in synovial tis-sues, it is likely that antiviral antibody signifi-cantly contributes to the high intrasynovial

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658 JOHNSON ET AL.

immunoglobulin concentrations found in CAEVinfection.The structural proteins of CAEV and closely

related visna-maedi, progressive pneumonia,and goat visna viruses have been well described(9, 10, 17, 30, 31, 35, 49). In this paper we reportthat the majority of the anti-CAEV humoralimmune response in experimentally arthriticgoats is directed toward two viral surface glyco-proteins, gp125 and gp9O. Antibody productionto these two antigens exceeds that to viral p28,the most abundant viral structural protein, by100- to 1,000-fold in serum and synovial fluidearly in viral infection. The host's immune re-sponse to CAEV gp125 and gp9O might have arole in the initiation and progression of thearthritis.

MATERIALS AND METHODSExperimental design. Fifteen goats were delivered

by caesarean section, given passive antibody transfer,and housed to ensure their lack of exposure to CAEV(2). Serum and synovial fluid samples from 10 goatswere taken 2, 6, and 9 months after intravenous andintraarticular inoculation with 105 8 50% tissue cultureinfectious doses of isolate 75-G63 ofCAEV (2), whichhad been triple cloned by limiting dilution in primarygoat synoviocyte cultures (27). Synovial fluid sampleswere taken from the inoculated joint. Controls includ-ed serum and pooled synovial fluid from five goatsreceiving tissue culture medium lacking virus. Anadditional six goats were inoculated by uncloned stockfrom the same viral isolate (106-2 50% tissue cultureinfectious doses) 38 months before sampling (2).

Radiolabeling of virus. Goat synoviocyte culturesgrown in 75-cm2 flasks were infected with clonedCAEV, deprived of methionine (minimal essentialmedium free of methionine and glutamine; Flow Labo-ratories, Inglewood, Calif.), and grown for 5 to 6 daysin the presence of 15 ml of a similar medium supple-mented by 2 mM glutamine with 160 LCi of [35S]me-thionine (specific activity, -990 Ci/mmol). Similarcultures were labeled with "4C-amino acids (250 ,uCiper flask) (9). Virus was purified from the medium bysucrose density gradient ultracentrifugation (9). Viruspreparations labeled with [35S]methionine and 14C-amino acids were compared on sodium dodecyl sulfate(SDS) gradient slab gels under reducing conditions, asdescribed below.

Surface labeling. Freshly collected, density gradient-purified CAEV (9) was surface labeled by lactoperoxi-dase-catalyzed (42 U/mg; Sigma Chemical Co., St.Louis, Mo.) iodination with 1 mCi of Na125I (17mCi/mg), using established methods (44) modified bythe addition of 25 RIx of freshly prepared 44 ,uM H202every 10 min during a 30-min incubation period. La-beled virus was 85 to 95% trichloracetic acid precipita-ble. Additional virus, partially disrupted by a prior 10-min, 37°C incubation in 0.5% Triton X-100, wasiodinated for comparison. There was a lack of signifi-cant 125j binding to virus in reaction mixtures withoutlactoperoxidase.

Galactosyl residues of viral surface glycoproteinswere labeled with tritium from sodium boro[3H]hy-dride after neuraminidase and galactose oxidase treat-

ment (40). A virus preparation not treated with galac-tose oxidase served as a control and failed toincorporate tritium.Bromelain treatnent of labeled virus. Lactoperoxi-

dase-iodinated or tritiated CAEV was treated withbromelain by previously described methodology (11).Undialyzed labeled virus containing 4 x 106 1251 cpmor 1 x 106 3H cpm was mixed with bromelain at 37°Cfor 12 h. The reaction mixture was then centrifuged at100,000 x g for 90 min and separated on a 20 to 45%sucrose gradient for 16 h at 30,000 x g. Fractions werecollected and compared with those of simultaneouslycentrifuged untreated radiolabeled material.

Immunoprecipitation of [35SJmethionine-labeled viralproteins. Sonicated [355]methionine-labeled virus wasdetergent disrupted as previously described (48). Theviral lysate was dialyzed against TEN buffer (20 mMTris, 1 mM EDTA, 100mM NaCl [pH 7.4]), brought to1% Nonidet P-40 and 0.1% SDS, and centrifuged at100,000 x g for 1 h immediately before use. Debrisfrom spent virus-infected cells, pelleted by low-speedcentrifugation from culture medium, was utilized forsome experiments and was treated in a similar manner.

Precipitation involved the sequential interaction ofvirus, goat antibody, affinity-purified rabbit anti-goatantibodies, and protein A-containing Staphylococcusaureus (Pansorbin; Calbiochem-Behring, La Jolla,Calif.). Rabbits were injected with goat IgGl, IgG2,and IgM (34), and rabbit anti-goat antibodies werepurified from serum by binding to a goat immunoglob-ulin-agarose matrix prepared by cyanogen bromidecoupling (16). Specific rabbit antibodies were eluted(41, 52) and immediately desalted. With 125I-labeledgoat IgGi or IgG2 (24) markers, 500 pl of rabbitantibody had a precipitating capacity of 100 ,ug (total)of IgGl and IgG2. Immunoglobulin concentrationswere determined by single radial immunodiffusion,using class-specific reagents as described previously(25).

Viral antigen (50,000 to 75,000 cpm; 2 to 4 ,ug ofprotein) (28) or infected cell pellet (450,000 cpm) wasincubated at room temperature in siliconized test tubesfor 1 h with goat serum or synovial fluid containing 100pg of IgGl plus IgG2. Rabbit anti-goat immunoglob-ulin was added, and 1 h later 100 RI1 of protein A-bearing S. aureus (10%o suspension) was added. Afteran additional hour, the precipitate was washed fourtimes with TEN buffer containing 0.1% Nonidet P-40,twice with TEN buffer containing 2 M NaCI, and oncewith TEN buffer alone (48). The washed staphylococ-cal pellets were resuspended in electrophoresis buffer(0.5 M Tris [pH 6.8], 2% SDS, 15% glycerol, 2.5% 2-mercaptoethanol), heated to 900C for 3 min, andcentrifuged. Supernatants were electrophoresed,treated for fluorography either with En3Hance (NewEngland Nuclear Corp., Boston, Mass.) or as de-scribed previously (5), and exposed to X-OMAT ARfilm (Eastman Kodak Co., Rochester, N.Y.) at -700Cafter drying under vacuum. The precipitated antigenswere compared with 14C-labeled protein standards ofknown molecular weights (MWs). Serum or synovialfluids from uninfected goats were used to assessbackground binding, and precipitations utilizing rabbitanti-p28 serum verified the disruption of viral antigens.The efficacy of the antibody-precipitating system wasconfirmed in each assay by the precipitation of >95%

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ANTIBODY TO SURFACE GLYCOPROTEINS IN CAEV 659

of the acid-precipitable "25I-labeled goat IgGl in thepresence of goat serum containing 100 ,g of IgGl andIgG2.CAEV p28 radioimmunoassay. The p28 for radio-

immunoassay was purified as previously described(43). Portions (20 ,ul) of p28 were iodinated with1,3,4,6-tetrachloro-3a,6a-diphenylglycuril (Iodogen;Pierce Chemical Co., Rockford, Ill.) (33). A total of80% of the radioactivity was trichloroacetic acid pre-cipitable; 35 to 40% could be precipitated by rabbitanti-CAEV, rabbit anti-CAEV p28, or goat anti-CAEVsera, with a background of 2% for nonimmune goat orrabbit sera. A single 28,000-MW band was seen onautoradiographs of iodinated material with SDS-poly-acrylamide gel electrophoresis (SDS-PAGE). Portions(60 to 120 ng) of "25I-labeled p28 (35,000 to 70,000 cpm)were reacted with fourfold serial dilutions of goatserum or synovial fluid and adequate serum from anon-CAEV-infected goat to ensure precipitation. After1 h at room temperature, rabbit anti-goat immunoglob-ulin serum was added, and incubation was continuedovernight at 4°C. After two washes with Tris-EDTA-saline buffer (0.2 mM Tris, 5 mM EDTA, 100 mMNaCl, 15 mM NaN3 [pH 7.6]) containing 5 mg ofbovine serum albumin per ml and 0.4% Triton X-100(45), the precipitates were counted. The dilution ofantibody at 50% antigen binding was determined fromthe linear portion of the sigmoid curve obtained byplotting percent binding versus log reciprocal dilution.Western blots. Portions (200 ,ug) of sucrose gradient-

purified CAEV were subjected to SDS-PAGE, asdescribed previously (50). Viral proteins were trans-ferred (8) onto nitrocellulose paper (0.2-p.m pore size;Schleicher & Schuell Co., Keene, N.H.) at 90 V for 23h at 4°C, using a Transphor electrophoresis unit(Hoeffer Scientific Instruments, San Francisco, Cal-if.). 14C-methylated protein standards were trans-ferred from adjacent lanes on the same gel. Aftertransfer, antibody binding to proteins on the paper wasdetected as follows: nonspecific binding sites wereblocked by a 1-h 37°C incubation in 10 mM Tris-0.99oNaCI (pH 7.4) (TS buffer) containing 5% bovine serumalbumin. Antigen-containing strips (5 mm, 16 stripsper gel) were incubated with 2 ml of sample diluted10-3 to 10-8 (serum) or 10-3 to 10-9 (synovial fluid)for 1 h at room temperature with constant agitation.The strips were rinsed six times with phosphate-buffered saline, twice with TS buffer containing 0.1%Nonidet P 40, and once with TS buffer alone. Afterthis, strips were incubated with 10 ml of rabbit anti-goat immunoglobulin serum (diluted 1:50) for 1 h, anda similar sequence was followed. The incubation of 106cpm of 125I-labeled purified staphylococcal protein A(Sigma) (24) took an additional hour. After a finalseries of rinses, the strips were sealed in plastic wrapand exposed to film.

Titration of antibody with 125I-surface-labeled virus.Gradient-purified CAEV was iodinated with lactoper-oxidase, dialyzed, and disrupted as described above.Serum and synovial fluid samples from two goats werediluted 10-fold from 100 ,ug of total IgG content;dilutions containing 100 to 0.01 ,ug of serum IgG orsynovial fluid IgG were reacted with 750,000 cpm ofiodinated viral antigen and then immunoprecipitatedas described above. After SDS-PAGE, gels were driedand exposed to film for 48 h.

RESJULTSComparison of [35S]methionine- and 14C-amino

acid-labeled CAEV proteins. To determinewhether [35S]methionine was incorporated intoall viral proteins, autoradiographs of SDS-PAGE gels of CAEV labeled with [35S]methio-nine or incorporating 14C-amino acids were

compared (Fig. 1). Polypeptides of similar MWswere present in both preparations, indicatingthat methionine was incorporated into all majorviral structural proteins, although some bandswere more intense with 14C labeling. ApparentMWs for the major CAEV proteins, indicated bythe arrows, are 125,000, 90,000, 70,000, 46,000,43,000, 28,000, 15,000, and 13,000.

Surface labeling of CAEV. Lactoperoxidase-catalyzed iodination of intact, freshly purifiedvirus was used to detect proteins occurring on

the surface of the virion. High-MW (125,000 and90,000) viral proteins were labeled and precip-itated by rabbit serum made against densitygradient-purified CAEV, as were 70,000- and15,000-MW structural proteins (Fig. 2, lanes Aand B). Additional wide bands of 45,000 and20,000 MW were labeled, although no similarbands were present in similarly purified 35S- or14C-labeled virus (Fig. 1) or after immunopre-

A B C

_K _

FIG. 1. Comparison by SDS-PAGE ofCAEV poly-peptides labeled with [35S]methionine (lane A) or 14C-amino acids (lane C). Lane B contains 14C-methylatedprotein standards of 200,000 (myoglobin), 92,500(phosphorylase b), 69,000 (bovine serum albumin),46,000 (ovalbumin), 30,000 (carbonic anhydrase), and14,300 (lysozome) MW. Virus labeled with each iso-tope (50,000 cpm) was applied to lanes A and C. Thearrows refer to viral proteins of 125,000, 90,000,70,000, 46,000, 43,000, 28,000, 15,000, and 13,000MW.

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A B C D E F G

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FIG. 2. Surface labeling of CAEV proteins andglycoproteins. Lanes A through E, from one gel, areautoradiographs of lactoperoxidase-iodinated virusand "C-labeled standards (lane E) used in Fig. 1; lanesF through I, from a second gel, are fluorographs ofvirus labeled with sodium boro(3H]hydride after treat-ment with neuraminidase and galactose oxidase. Io-dinated viral surface proteins (lane A) were comparedwith standards (lane E) used in Fig. 1. The iodinatedviral surface proteins were compared with the samematerial immunoprecitipated by rabbit anti-CAEV se-rum (lane B). Treatment of virus with Triton X-100before labeling results in the pattern present in lane D,and immunoprecipitation of this antigen by rabbit anti-CAEV serum gives the pattemn present in lane C.Tritium incorporation into viral glycoproteins wasdemonstrated in rabbit anti-CAEV serum immunopre-cipitates (lane F) and unprecipitated antigen (lane I),whereas nonimmune rabbit serum failed to precipitateany activity (lanes G and H).

cipitation of 35S-labeled cell culture-associateddebris. Therefore, these bands may representnonprotein contaminants of the viral preparationwhich may be of host origin. Additional viralproteins were labeled when CAEV was disrupt-ed with Triton X-100 before the lactoperoxidase-catalyzed iodination (Fig. 2, lane D); immunerabbit serum interacting with this material (laneC) precipitated bands similar in MW to the majorproteins present in [35S]methionine- or 4C-ami-no acid-labeled virus. Tritium was incorporatedinto viral surface galactosyl residues after neur-aminidase and galactose oxidase treatment (Fig.2). The three high-MW bands (125,000, 90,000,and 70,000; lane I) were labeled, demonstratingtheir glycoprotein nature, and were precipitatedby CAEV-immune rabbit serum (lane F). Somesugar residues were present in the 15,000-MWprotein as well. Label was not incorporated intoviral proteins by either procedure in the absenceof the enzyme (data not shown).

Bromelain treatment of iodinated virus. Toconfirm the surface location of the iodinated

proteins, CAEV was placed on 20 to 45% su-crose gradients either directly after lactoperoxi-dase iodination or after iodination, bromelaintreatment, and centrifugation. Enzyme-treatedvirus lost the majority (>80%) of its radioactiv-ity and shifted to a greater density (from 1.15 to1.16 g/cm3) (Fig. 3). Electrophoresis of 125I-labeled virus demonstrated some iodine incorpo-ration into viral p28 (see Fig. 7, lane E), indicat-ing inadvertent disruption of some virions duringpurification and minor labeling of internal pro-teins, accounting for the 20% label retained afterbromelian treatment. The low-density radioac-tivity present on sucrose gradients of iodinatedCAEV not treated with bromelain was probablyeliminated in the centrifugation step used toconcentrate bromelain-treated material. Compa-rable data resulted when tritiated virus under-

1:

101

9

1.15

11.16

Fraction Number

FIG. 3. Effect of bromelain treatment on the densi-ty and presence of lactoperoxidase-iodinated polypep-tides of CAEV. The pattern of 125I incorporation intoviral polypeptides of this material is shown in Fig. 7,lane E. Undialyzed labeled virus (-) or virus treatedwith bromelain and centrifuged to concentrate it (Owas repurified on 20 to 45% sucrose density gradients.The distribution of 1251 in gradient fractions of thesetwo preparations is demonstrated, and the density of

peak fractions is recorded above them.

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went a parallel procedure (data not shown),although in this instance more than 95% of theradioactivity was lost after bromelain treatment.Immunoprecipitation of [35S]methionine-la-

beled CAEV. Figure 4 shows the ability of serumand synovial fluid from an infected goat toprecipitate [35S]methionine-labeled virus 6months after infection. The most visible bandswere viral gp125 and p28, with the lesser bandsbeing gp9O, p15, and p13. Sera and synovialfluids from all nine goats precipitated gp125,gp90, p28, and p15; in addition, those from someof them precipitated p13. Pooled synovial fluidor serum from five mock-infected goats had noprecipitating activity, and rabbit serum specificto CAEV p28 precipitated only p28 (data notshown).When spent cellular debris pelleted from vi-

rus-infected cell culture medium replaced puri-fied virus as the radiolabeled antigen, the im-munoprecipitation patterns were unaltered (Fig.5), indicating that goat antibody recognized nomajor antigens present in spent virus-infectedcells that were not themselves incorporated intothe virion.

Viral p28 radioimmunoassay. Despite the abili-ty of all infected goats to precipitate 35S-labeledviral p28, quantitative antibody titers to thisantigen remained low in both serum and synovi-

A B C D

X aw. *

FIG. 4. Immunoprecipitation of [35S]methionine-labeled CAEV by serum (lane A) and synovial fluid(lane B) of a virus-infected goat 6 months after inocula-tion. Data are presented in comparison with 75,000cpm of "5S-labeled viral antigen (lane C) and 14C-methylated protein standards (lane D; see the legendto Fig. 1). After immunoprecipitation as described inthe text, samples were electrophoresed on an SDS-PAGE gel under reducing conditions. The gel was thentreated for fluorography, dried, and exposed for 6weeks (lanes A and B) or 2 weeks (lanes C and D).

A B C D

4,

FIG. 5. Immunoprecipitation of [35S]methionine-labeled infected cell debris. The antigen came fromlow-speed centrifugation of pellets of cell culture me-dium from which the virus in Fig. 4 was purified, usingconditions identical to those described in the legend toFig. 4 and a 2-week exposure of film. The antigen (laneB) was compared with "4C-methylated protein stan-dards (lane A) and two representative immunoprecipi-tation patterns (lanes C and D). Arrows mark the topsof the stacking and resolving gels.

al fluid of the animals for the first 9 months afterinfection, with only a moderate increase in titerswhen a second group of goats was tested 38months postinoculation (Table 1). Although arange of values was evident, anti-p28 titers werebelow 1:400 for the first 6 months after infection,with mean titers of 1:182 (synovial fluid) and1:67 (serum). Antibody activity was quite simi-lar in serum and synovial fluid samples, inconstrast to the considerably higher immuno-globulin content of the synovial fluid early ininfection. At 6 months postinoculation 0.1 to 4mg of IgG was needed for 50% precipitation of60 to 120 ng of antigen; therefore, it appearsunlikely that p28 antibody accounted for a largeproportion of the intrasynovial immunoglobulinpopulation.Western blots. In an effort to determine which

viral proteins were the focus of the goat's im-mune response, 10-fold serum or synovial fluiddilutions from 10 virus-infected and 5 controlgoats were incubated with nitrocellulose stripsto each of which approximately 18 Rxg of viralantigens had been transferred. The goats hadbeen experimentally infected for 6 months. Typ-ical titration profiles of two sera and two synovi-al fluids from infected goats are presented in Fig.6. The major antigens recognized by the goats athighest dilutions were gp125 and gp9O. A wideband of antibody binding was also present in the45,000-MW region of the strip. Antibody bindingto p28 and p15 was evident in some samples at

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TABLE 1. Anti-CAEV p28 antibody titers after experimental infectionTiter" in: Serum IgG/syno-

Time postinoculation vial fluid IgGb(mo) Serum Synovial fluid (mg/ml)

2c 15 ± 5 (2-61) 13 + 5 (0.2-63) 8.3/21.06c 66 ± 10 (14-300) 117 ± 12 (12-400) 15.8/47.39c 67 ± 7 (14-155) 182 + 20 (9-1,220) 14.7/25.3

38d 590 ± 19 (26-1,000) 960 + 33 (130-2,800)a Reciprocal dilution precipitating 50%o of th- immunoprecipitable counts of 60 to 120 ng of 125I-labeled CAEV

p28. Results are expressed as mean ± standard error of the mean; the range is given within parentheses.b IgG concentrations were calculated by adding the IgGl and IgG2 concentrations determined by single radial

immunodiffusion.C n = 9 goats.d n = 6 goats.

lo-,, the lowest dilution tested, but not in thoseshown in the figure. Antibody to gp9O wasalways present at high sample dilutions (10-4 to10-6) and could be detected at a 100- to 1,000-times-greater dilution than antibody to p28. In 8of 9 sera and 9 of 10 synovial fluids examined,

F G

&

FIG. 6. Western blots titrations with CAEV-im-pregnated nitrocellulose. Dilutions (10' to 10-8 forserum or 10-3 to 10-9 for synovial fluid) were incubat-ed with nitrocellulose strips to which CAEV proteinshad been electrotransferred. Lanes A through D re-ceived antigen from one SDS-PAGE gel, and lanes Ethrough H received antigen from another, with thestandards from that gel. Lane A, 1:100 dilution ofserum from an uninfected goat; lane B (six strips), 10-fold dilutions of infected goat serum showing anti-gp9Oactivity to 104 and anti-gp125 activity to 103; lane C,synovial fluid (seven strips) of the same goat withtitrations endpoints of 105 and 104, respectively, forgp9O and gp125; lane D, 1:50 dilution of rabbit anti-CAEV p28 serum, directly treated with '25I-labeledstaphylococcal protein A; lane E, reaction of anti-CAEV hyperimmune goat serum at a 1:50 dilution;lanes F and G, dilutions of serum and synovial fluid(seven and six strips, respectively) from another in-fected goat; lane H, 14C-methylated standards. Onlythe 69,000- to 14,300-MW standard proteins are visibleon this autoradiograph (see the legend to Fig. 1 for adescription of standards).

the gp9O titer was 10 times greater than thegp125 titer; in other instances, the two had equaltiters. In 5 of 10 instances, more anti-gp9Oactivity was present in synovial fluid than inserum. In the other five cases, where the end-points were similar for the two samples, theautoradiographic line for synovial fluid was of-ten of greater intensity than that for serum.Nitrocellulose strips incubated with serum ofcontrol goats, diluted 1:100 (Fig. 6, lane A), orwith synovial fluid pools from controls, diluted1:100, did not retain 125I-labeled protein A.Incubation with 1:50 dilution of anti-CAEV se-rum of goat origin, under identical conditions(Fig. 6, lane E), revealed that the major viralantigens were immunologically reactive, andanti-p28 rabbit serum reacted with that proteinas well (lane D). The calculated IgG content atdilutions where the 90,000-MW antigen was lastvisible ranged from 0.02 to 11 ,ug. When calcula-ble, the amounts of IgG present in samplesreacting with p28 were 0.1 to 2 mg.

Titration of goat antibody with 125I-labeledvirus. Viral antigens radioiodinated by lactoper-oxidase treatment (Fig. 7, lane E) were immuno-precipitated by dilutions of serum and synovialfluid from goats infected for 6 months to confirmdata obtained from Western blots. Ten-fold se-rum dilutions containing 100 to 0.1 ,ug of IgG andsimilar dilutions of synovial fluid containing 100to 0.1 ,ug of IgG were reacted with labeledantigen (Fig. 7). The precipitating activity ofthese samples for CAEV gp9O was presentthroughout this dilution range. Anti-gp125 activ-ity was partially lost at 0.1 ,ug of IgG in serumand 0.01 pg of synovial fluid IgG, whereassynovial fluid from an uninfected goat had mini-mal precipitating activity even at 100 ,g ofimmunoglobulin.

DISCUSSION

CAEV, labeled with 14C-amino acids or[35S]methionine, contained a series of high-MWglycoproteins (125,000, 90,000, and 70,000 MW,

A B c D ErF

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all located on the virion surface), a relativelyabundant nonglycosylated protein of 28,000MW, and two low-MW proteins (15,000 and13,000 MW). These findings are in generalagreement with other reports for goat lentivi-ruses (9, 10, 17, 49) and are similar to proteinlabeling patterns for ovine lentiviruses, visnavirus, and progressive pneumonia virus (7, 10,17, 21, 30, 31, 35).Glycosylation of the 90,000-MW protein of

CAEV is unreported, but previous studies dem-onstrating glycosylation of CAEV proteins ofabout 135,000 and 70,000 daltons relied on theincorporation of [3H]glucosamine into the virion(9, 10). Failure to detect this surface glycopro-tein may be due to the paucity of glycosamine inthe CAEV gp9O; in addition, tritration of galac-tosyl groups amplified by neuraminidase treat-ment is undoubtedly more sensitive. The glyco-sylated 15,000-MW surface protein mayrepresent a piSE counterpart (51) in the lenti-virus subgroup. A 12,000-MW envelope glyco-protein and a low-MW product of the env genehave been described for visna virus (32, 52).

Six months after viral infection, goats hadantibodies to four or five of the six major CAEVproteins: gp125, gp9O, p28, and p15 were recog-nized by all goats; some animals precipitated p13as well. There were no qualitative differences inthe antiviral antibody present in serum andsynovial fluid. Goat antibodies detected no addi-tional [35S]methionine-labeled proteins in cellu-lar debris from virus-infected culture medium,reducing the likelihood that antigens not incor-porated into the virion (i.e., separate virion-coded cell surface antigens or host cell proteins)are important to the immunological response ofthe goats.Because p28, the most abundant protein in the

virion (9), is recognized by most virus-infectedgoats (1, 43), it was important to quantitivelyassess its role in the immune response. Radio-immunoassay titers in serum and synovial fluidto p28 remained low for months after infection,in the face of active arthritis and massive intra-synovial IgGl production (25).'However, theresults correlate well with a relative lack of viralantigen expression even early in the genesis ofCAEV arthritis (2) and with the paucity ofimmunofluorescent staining of p30 in visna vi-rus-infected choroid plexus (22). Whether lowantibody titers to CAEV p28 reflect a lack ofexpression of this viral protein (6) or whether theimmune system of the goats remains shieldedfrom the antigen by the internal budding of virusparticles (13, 37) remains unsolved.Antibody to gp9O could be detected at dilu-

tions as great as 10-6 with Western blots, andtitration endpoints corresponded to IgG concen-trations of 0.02 to 11 ,ug in the reaction mixture.

A B C D E F G H I J K L

a

FIG. 7. Immunoprecipitation of 125I-labeled sur-face polypeptides of CAEV. Serum or synovial fluidwas obtained from goats at 6 months postinoculation.Tenfold dilutions of goat serum (containing 100 to 0.1,Lg of total IgG) was reacted with 750,000 cpm oflactoperoxidase-iodinated intact CAEV (lanes Athrough D). Dilutions of synovial fluid from the samegoat (100 to 0.01 Fg of total IgG) interacted with thesame antigens (lanes G through K), whereas 100 ,g ofIgG from an uninfected goat precipitated the pattern inlane L. Labeled antigen (50,000 cpm) is present in laneE. Material was electrophoresed under reducing con-ditions as described in the text; the gel was dried andexposed to film for 48 h.

Immunoprecipitation of lactoperoxidase-iodin-ated virus confirmed that anti-gp9O activity waspresent in serum diluted to an immunoglobulincontent of 0.1 ,ug and in synovial fluid diluted toat least 0.01 ,ug. In contrast, when p28 bindingcould be observed with Western blots, IgGcontents of 0.1 to 2 mg were calculated, a figurecomparable to the range of 0.1 to 4 mg present at50% titration endpoints in the radio-immunoassay, suggesting that the two tests areof comparable sensitivity. Discussion of therelative antibody binding to nitrocellulose-bound gp125, gp9O, and p28 is difficult, due todifferential denaturing of antigens during SDS-PAGE and to the difficulty in quantitively trans-ferring high-MW proteins from polyacrylamidegel onto nitrocellulose paper (20). In this particu-lar instance, however, these arguments increasethe confidence that considerable anti-gp125 andanti-gp9O antibody is present. The presence of100- to 1,000-fold more antibody to gp9O than top28 and the relatively high anti-gp125 titersindicate that these antigens form the major focusof the host's antiviral immune response andimplicate antibodies to them as contributors tohigh intrasynovial IgGl concentrations.How this differential immune response to viral

antigens comes about is an important question.

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664 JOHNSON ET AL.

Observations on visna virus indicate that gp135is exported to the extracellular fluid at a highrate, whereas gag gene products are retainedwithin the cell (52). If processing of CAEV issimilar, high-MW proteins may be extruded intothe synovial fluid or may occur independently ofother viral proteins on the surface of virus-infected cells, accounting for the disparity inantibody titers. In murine leukemia virus infec-tion, gp70 expression may occur independentlyof virion maturation (15, 26). Alternatively, thepresence of antiviral antibody in the synoviummay result in the shedding of coreless aggregatesof virion surface proteins into the synovialspace, similar to the process that occurs whenvariant-specific neutralizing sheep antibody isadded to visna virus-producing cell cultures (19).Sheep infected with visna virus react to high-

MW antigens by immunoprecipitation (32) andimmunodiffusion (7) tests, and other observa-tions indicate that anti-gp135 antibody is respon-sible for the functionally high levels of neutraliz-ing activity that are characteristic of the disease(46). It is intriguing that virus-neutralizing activi-ty was not demonstrated in the sera of goatsinfected with CAEV (27), although, as demon-strated here, goat serum can react with viralantigens, which should stimulate neutralizingactivity. The relationship between CAEV gp125and gp90 is unclear. Although proteins of similarMWs packaged in the mature visna virion areunrelated to each other (52), the same workersfound a partially glycosylated intracellular90,000-MW protein that was probably related togp135, and they hypothesized that a second85,000-MW glycoprotein represented a break-down product that could conceivably be incor-porated into the virion.

Exploration of the antiviral immune responseby goats actively developing CAEV arthritisindicated a preferential synthesis of antibody totwo virion glycoproteins, gp125 and gp9O. Al-though infected goats responded immunological-ly to the abundant virion core antigen p28, twotechniques indicated that the relative binding ofantibody to this antigen was about 100 to 1,000times less than that to surface glycoproteins.Synovial fluid antibody activity to gp125 andgp9O could be diluted to the range of 10 to 100ng, indicating that these antibodies contribute tothe high intrasynovial immunoglobulin concen-trations observed in the disease.

ACKNOWLEDGMENTSWe thank Scott Adams for supplying some of the synovial

fluid samples and Alberta Brassfield and Deta Stem for experttechnical assistance. We also thank Phil Cheevers for hiscritical review of the manuscript.

This work was supported by Public Health Service grantAM 27680 and Immunology Training grant AI 07025 from theNational Institutes of Health and by the Agricultural ResearchCenter of Washington State University (project no. 0432).

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